Cancer is currently treated primarily with one of the following three types of therapies: surgery, radiation or chemotherapy. Frequently a combination of two or more of the therapies are prescribed in order to optimize the probability of a successful outcome.
Surgery is the traditional approach whereby all or part of a tumor mass is removed. Surgery is typically only effective for treating cancers in their earlier stages. Surgery is also limited to localized masses that are accessible to the surgeon and are not disseminated cancers like leukemia for example. Statistics have shown that, for more than 50% of cancer patients, by the time they are diagnosed surgery is no longer an effective treatment. Surgical procedures are also thought to increase the chances of metastases by dislodging small colonies of cells into the bloodstream. Most cancer patients do not die from surgery but rather from the subsequent metastasis and recurrence of cancer.
Radiation, much like surgery, is effective when the cancer is diagnosed in the early to mid stages and the disease is localized in a defined region of the body. This allows a maximal dose to be focused on the proliferative tissue while minimizing the exposure to adjoining normal tissue. In practice it is extremely difficult to shield the nearby normal tissue from the cytotoxic effects of the radiation and still deliver a therapeutic dose. An additional complication of radiation is the induction of radiation resistant cells during the course of treatment. Thus even the best radiotherapeutic techniques often result in incomplete tumor reduction and subsequent recurrence.
Chemotherapy has historically been designed to attack either rapidly dividing cells or cell metabolism. Based on its ability to permeate throughout most body tissues, it holds, in theory at least, the ability to address metastases. Although it can be effective, the side effects from these toxic compounds can be severe, e.g., vomiting, hair loss, weight loss, and immune system suppression through depleted white blood cell counts. Because of the severe side effects, many patients cannot successfully complete the entire cycle of treatment. Some cancer patients even die from the chemotherapy-induced side effects.
Chemotherapy in spite of these obstacles achieves complete remission in many patients. Based on available means of assessing the cancer burden in the body, they appear to be cured. However, a high percentage of these same patients experience relapse and death due to the cancer. For the individual patient there are many mechanisms which may ultimately render the chemotherapeutics ineffective. Chief among them are the pharmacokinetics which are manifested in bioavailability and distribution and the pharmacodynamics which are manifested in the mechanism of cell death and drug resistance. Furthermore, the recent discovery of cancer stem cells and their proposed role in driving and maintaining tumor growth and metastasis, adds one further level of complexity to the problem.
Bioavailability is the percentage of an administered dose of unchanged drug which reaches the systemic circulation. While the ease of administration of oral cancerostatic drugs is extremely attractive, the bioavailability is typically very low and inconsistent from person to person. This is due to the fact that the absorption of anticancer drugs in the gastrointestinal tract is incomplete and the drugs themselves can be chemically modified to an inactive form. Once in the bloodstream, the drugs are immediately confronted with hepatic first pass metabolism in addition to toxic removal systems such as glutathione-S-transferase. Oral administration therefore makes dosing calculations very difficult and targeting typically nonspecific. While much research is currently focused on oral, oral buccal, oral sublingual and transdermal drug delivery systems, intravenous administration is often necessary in order to approach 100% bioavailability of cancerostatic drugs.
Distribution is the dispersion or dissemination of substances throughout the fluids and tissues of the body. Many chemotherapeutics upon entrance to the systemic circulation become evenly distributed and based, on their mechanism of action, begin to work on both normal and cancer cells. This nonspecific, nontargeted delivery results in the typical severe side effects and thus limits the dosing to a rather small therapeutic window. Recent advances like drug encapsulation in antitumor liposomes, attachment to albumin particles, and antibody ligand surface binding have improved distribution and targeting issues. In particular, antitumor liposomes administered by (IV) (intravenous) provide high bioavailability, encapsulation protection from metabolism, long systemic circulation times due to pegylation, and passive and active targeting through EPR (Enhanced Permeation and Retention) and surface decoration [1, 2].
The mechanism of action for the vast majority of currently available anticancer drugs is through cell division inhibition, by acting on either DNA synthesis or function. By design, these treatments attack rapidly dividing cells. This results in the nonspecific killing of rapidly dividing normal cells as well as cancer cells and weakens the immune system. While several recent advances such as antiangiogenic compounds and kinase inhibitors hold promise, metastases are still largely incurable. Recently however, much interest has been focused on the normal physiological process of apoptosis, also known as programmed cell death. By selectively inducing this cell death mechanism in cancer cells, it may be possible to control and eliminate tumors [3, 4].
Drug resistance is typically classified as either inherent or acquired. The Goldie-Coldman hypothesis estimates that 1 in every 1,000,000 cancer cells is resistant to anticancer drugs due to inherent rates of mutation. This form of inherent drug resistance is developed before any exposure to an anticancer drug has taken place. Acquired drug resistance can be developed by the sublethal exposure of cells to anticancer drugs. This form of drug resistance is caused by the upregulation of cellular defense mechanisms. It is well known that levels of the major cellular antioxidant, GSH, is elevated in cancer cells to help buffer the high levels of reactive oxygen species (ROS). It is also well known that GSH is a strong detoxifier of anticancer drugs. Recently the protective effect of GSH against ionizing radiation has also been demonstrated in cancer stem cells [5]. The use of cancerostatic natural product may have the advantage of being an effective anticancer agent while not inducing drug resistance.
Apoptosis in normal cells can be induced by both extrinsic and intrinsic factors. Regardless of the method of induction, the sequence of events must culminate in the activation of the caspases. Extrinsic apoptosis is death receptor mediated while intrinsic apoptosis is mitochondria mediated and characterized by the rapid release of cytochrome c into the cytosol. The activation of either of these pathways by apoptotic stimuli is exceedingly complex. Attempts at trying to integrate the complexity of signaling events with the regulation of apoptosis has resulted in a dizzying array of possibilities which may or may not be physiologically significant. In spite of this complexity, the concept of perhaps cancer cell specific apoptosis remains a viable option [4].
Apoptosis in cancer cells is further complicated by the fact that they have broken apoptotic machinery. Parts of the process are either missing or functionally mutated, with the most notable being the tumor suppressor protein, p53. While at least 50% of cancers demonstrate either nonexistent or nonfunctional p53, cancer cells also contain various and numerous other apoptotic defects. This is consistent with the fact that cancer cells contain a high degree of cell to cell heterogeneity. Fortunately cancer cells can still undergo apoptosis and furthermore can be sensitized to do so through oxidation-reduction modulation [6].
Apoptosis has been widely reported to be modulated by changes in the oxidation-reduction state of the cell. Although the exact mechanism of action has not been elucidated, GSH and ROS have been strongly implicated. Levels of ROS are higher in cancer cells than in their normal counterparts [6]. Levels of GSH are also higher in cancer cells, by as much as 100% over those found in normal cells. Since excessive levels of ROS are toxic to cells, cancer cells with inherently higher levels of ROS should be more sensitive to further insult. However, evidence demonstrates by increasing the oxidative stress in cancer cells by adding ROS generators that apoptosis is not always induced [7].
GSH depletion has also been associated with the onset of apoptosis [7, 8, 9, 10]. Recent work has shown that GSH depletion is indeed necessary for the progression of apoptosis activated by both extrinsic and intrinsic pathways in cancer cells. Apoptosis by GSH depletion was found to be independent of ROS formation in cancer cells [7]. Depletion of GSH to levels approaching 5% of the controls in cancer cells leads to approximately 85% of the cells undergoing apoptosis. Therefore, compounds capable of depleting GSH levels in cancer cells should induce and/or sensitize cells to apoptosis.
Any protocol which is proposed for the chemotherapeutic treatment of cancer cells need to address the impact on cancer stem cells. Cancer stem cells are not easy to kill. Recent studies have found that cancer stem cells are particularly resistant to ionizing radiation. In addition, those cancer stem cells which contain relatively low levels of ROS were much more resistant to ionizing radiation compared to those with higher levels. Further study showed that the highly resistant cancer stem cells contained higher levels of GSH [5]. Thus as is the case in ordinary cancer cells, depleting GSH in cancer stem cells might induce or sensitize the cells to apoptosis.
Agents which deplete GSH selectively in cancer cells will have a significant effect on apoptosis. In order to achieve high levels of apoptotic induction GSH must be taken to very low intracellular levels, approaching 0-5% of the endogenous concentrations. Therefore, while cancer cell apoptosis can be achieved by significant GSH depletion alone, an additional cancerostatic agent is desirable to ensure the desired outcome.
A number of different approaches exist for reducing or depleting intracellular GSH. The primary mechanisms involve: 1) tying up GSH by forming a chemical complex between GSH and an electrophilic agent, 2) introducing an enzyme inhibitor to prevent GSH synthesis, and 3) raising GSH levels in order to activate enzyme feedback inhibition.
Dicarbonyl compounds have been studied for over 50 years because they possess cancerostatic properties at relatively low concentrations [11]. Of particular interest is the dicarbonyl compound, methylglyoxal (also known as pyruvaldehyde). Containing just a (3) carbon backbone, it is an extremely small, water soluble, and highly reactive compound. Due to its size and reactivity as an electron acceptor, it is a unique chemical and biochemical compound. Of critical importance are the following facts: 1) methylglyoxal is a naturally occurring metabolite in living systems, 2) methylglyoxal is catabolized to D-lactic acid by the glyoxalase enzyme system consisting of glyoxalase I and glyoxalase II, 3) methylglyoxal forms a nonenzymatic hemithioacetal adduct with GSH, and 4) methylglyoxal in the absence of glyoxalase II activity causes GSH to become trapped in the glyoxalase I intermediate S-lactoylglutathione thus depleting GSH levels.
Albert Szent-Gyorgyi in the 1960's, advanced the hypothesis that methylglyoxal might act as a natural brake on cell division by keeping cells in the resting state [12]. Subsequent work by his team and others showed that methylglyoxal treatment of cancer cells at levels of approximately 1-3 mM caused protein synthesis inhibition, arrested cell growth, and induced apoptosis [13]. Normal cells were not affected. These findings and much additional research over the intervening 40 years have supported and expanded this work. Of particular significance is the recent work of Manju Ray. She and her team have enhanced the understanding of the action of methylglyoxal at the cellular level, studied and reported the pharmacokinetics and toxicity, and conducted human cancer clinical trials [14, 15]. Additionally, there are a number of worldwide anecdotal cases of people self-administering methylglyoxal to treat cancer.
The majority of the early studies involving methylglyoxal and its cancerostatic action were conducted either with cultured cancer cells or with mice that had been innoculated intraperitoneally with cancer cells. This work consistently and repeatedly reported that concentrations approaching 3 mM were required to achieve 95-100% cell death. Methylgloxal administered orally cannot achieve these levels (216 mg/kg) in mammals based on pharmacokinetic bioavailability data. (IV) administration of methylglyoxal at these levels has not been reported.
Delivery of methylglyoxal to the bloodstream by any means results in its rapid catabolism. Pharmacokinetic studies in mice have shown that a single oral dose of methylglyoxal of 200 mg results in a peak blood concentration of approximately 20 nmol/cc or 0.02 mM at 4 hours with total clearance after 12 hours [16]. These levels are significantly lower than the 1-3 mM reported to induce significant cancer cell death. The presence of high levels of GSH and a full complement of glyoxalase enzymes makes delivering and maintaining pharmaceutical levels of methylglyoxal in the bloodstream unattainable. It is likely this is the reason that the limited number of previous clinical trials utilizing methylglyoxal to treat cancer have reported mixed results. Liposome systems have become a popular drug delivery platform for a number of important reasons [1, 2]. First, liposomes are composed of naturally occurring lipids which make them nontoxic and biodegradable. Second, liposomal drug encapsulation protects the active ingredients from the metabolic action of the body thereby preventing degradation and dilution. Third, liposomes can be modified to control their drug release rates. Fourth, in cancer, liposomes can be designed to migrate and preferentially accumulate at tumor sites as a result of their ability to extravagate through the large pores in the capillary endothelium. Thus liposomes may be custom designed for specific drug delivery needs by varying a number of critical variables including membrane chemical composition, particle size, surface treatment, and charge.
Prior artisans have explored a number of avenues in order to induce apoptosis in cancer cells by trying to deplete intracellular GSH levels. Such avenues have included the use of biologically active whey protein [17, 18], N-acetylcysteine [19], buthionine sulfoximine [20, 21, 22, 23, 24], and GSH complexing agents such as cinnamaldehyde [25], curcumin [26], quercetin [27], and isothiocyanates [28]. Results have demonstrated improvements in cancer patient survival times but not nearly to the extent necessary to be described as significant. Likewise recent chemical studies with oral methylglyoxal have increased survival rates and even resulted in some remission [14]. But again results have been variable. Recognition of challenges with delivering therapeutic levels of methylglyoxal have led researchers to form chemical conjugates to protect methylglyoxal in the bloodstream or introduce glyoxalase I inhibitors but also with less than hoped for results.
What is lacking in the art is: (1) a systemic therapy that can be used in conjunction with either surgery or radiation, (2) a therapy that leverages normal cellular physiological processes, (3) a therapy that can be given in nominal doses with compounds that, if they do come into contact with normal cells, are quickly metabolized to nontoxic substances, and (4) a therapy that causes little to no side effects in the patient.
The inventors of the current patent have research experience with both methylglyoxal and GSH dating back more than 30 years. Methylglyoxal was found to be missing in proliferative tissue but present in the organized tissue of origin [29] and GSH exhibited the opposite trend [30]. The latter studies also demonstrated the efficacy of using buthionine sulfoximine to lower the concentrations of GSH with biological consequences.